Cardiovascular diseases including atherosclerosis increase gradually as adult disease is growing. Atherosclerosis frequently occurs in cerebral artery or in coronary artery. Cerebral atherosclerosis carries headache, dizziness and mental disorder, and is a cause of cerebral infraction. Coronary arteriosclerosis causes pain and arrhythmia in the heart, leading to angina pectoris or myocardial infraction. Such diseases further cause hypertension, heart disease, cerebral hemorrhage, etc, making atherosclerosis related diseases the leading cause of death of men at the age of 50˜60 s.
High blood cholesterol causes coronary cardiovascular diseases. In order to reduce blood cholesterol, inhibition of enzyme involved in lipid metabolism or a dietary treatment designed to limit the intake of cholesterol and lipid has to be enforced.
For the purpose of preventing such diseases, attempts have been made to reduce low-density lipoprotein (LDL) by inhibiting cholesterol absorption and biosynthesis thereof (Principles in Biochemistry, lipid biosynthesis, 770-817, 3rd Edition, 2000 Worth Publishers, New York; Steinberg, D. et al. N. Engl. J. Med., 320: 915-924, 1989).
The production of LDL oxide in blood has been the subject of study since it is regarded as the cause of atherosclerosis (Circulation, 91: 2488-2496, 1995; Arterioscler. Thromb. Vasc. Biol., 17: 3338-3346, 1997). In particular, a recent report saying that the production of foam cells is resulted from the inflow of HM-LDL (highly modified LDL), generated by peroxidation and structural transformation of LDL, into macrophages triggered the study on the mechanism of the production and elimination of LDL peroxide (Curr. Atheroscler. Res., 2: 363-372, 2000).
Plague formation and breakage in inside of vascular wall lead to myocardial infraction, and chronic inflammation on vascular wall by the damage of it results in atherosclerosis, which is believed to be a rather defense mechanism than damage mechanism (Circ. Res. 89: 298-304, 2001).
Acyl-CoA: cholesterol acyltransferase (ACAT) is an enzyme that esterifies cholesterol and its working mechanism is in act in three regions of body (intestine, liver and vascular wall cells).
First, ACAT esterifies cholesterol and then helps the absorption of cholesterol in intestines. Second, cholesterol which is taken in from outside or produced in inside of body is accumulated in a carrier named VLDL (very low-density lipoprotein) in the liver, which is then provided to each organ of body through blood vessels. At this time, cholesterol is converted into cholesteryl ester by ACAT, enabling the accumulation of cholesterol in a carrier. Third, ACAT esterifies cholesterol in arterial wall cells, promoting the accumulation of cholesterol in cells, which is a direct reason for atherosclerosis.
By the activity of ACAT, foam cells include a huge number of cholesterol ester that is induced from cholesterol. Thus, the formation of foam cells induced from macrophages and smooth muscle cells is very important in experimental and clinical aspects. The growth of foam cells in vascular wall is directly related to the increase of ACAT activity. Therefore, an ACAT inhibitor might be effectively used as a powerful anti-atherosclerotic agent.
Therefore, an ACAT activity inhibitor has to and is expected to (1) reduce cholesterol taken in by inhibiting the absorption of cholesterol in intestines, (2) reduce blood cholesterol by inhibiting the release of cholesterol into blood vessels and (3) prevent atherosclerosis by inhibiting the accumulation of cholesterol in vascular wall cells.
All the ACAT activity inhibitors reported as of today are the inhibitors of the activity of mouse liver microsomal ACAT or mouse liver macrophage (J774) ACAT. Human ACAT is divided into two types; ACAT-1 and ACAT-2. Human ACAT-1 (50 kDa) works largely in the liver, adrenal gland, macrophage and kidney of an adult, and human ACAT-2 (46 kDa) works in the small intestine (Rudel, L. L. et al., Curr. Opin. Lipidol. 12: 121-127, 2001). The inhibition of ACAT activity has been a useful strategy for the prevention and the treatment of hypercholesterolemia, cholesterol gallstones or atherosclerosis owing to its mechanisms of inhibiting the absorption of cholesterol taken in from food and inhibiting the accumulation of cholesteryl ester in vascular wall (Buhman, K. K. et al., Nature Med. 6: 1341-1347, 2000).
Probucol, N,N′-diphenylenediamine, BHA (butylatedhydroxyanisol) and BHT (butylated hydroxy toluene), synthetic phenols used as anti-oxidatant agents, that have been used for the treatment of hyperlipidemia, reduce LDL cholesterol, weaken LDL-oxidation and reduce the lesion formation, showing excellent anti-oxidative activity but carrying serious side effects, so that they are limited in use.
Therefore, the treatment of patients with hyperlipidemia or atherosclerosis with a LDL anti-oxidative agent together with a lipid lowering agent is promising.
In the meantime, T. nucifera is a kind of evergreen needle-leaf tall tree belonging to Taxaceae, which is only distributed in Korea and Japan. T. nucifera is an edible, ornamental, medicinal and industrial plant. Its seeds have been eaten or produced as oil. And, its fruits have been used for the treatment of extermination, hair-regrowth, stomach-strengthening, and intestinal hemorrhage, especially in Chineses medicine and folk remedies, and the wood itself has been used for construction, making facilities and making ship (Kim, T. J., Plant Resources in Korea I, p40, Seoul National Univ. Press, 1996; Yook, C.-S., Illustrated of Flora of Asia, p23, Kyungwon Publishing Company, 1997). Ingredients of T. nucifera, separated from its leaves and seeds, are sesquiterpenoids (Sakai, T. et al., Bull. Chem. Soc. Japan, 38: 381, 1965), labdanes, abietanes including diterpenoids (Sayama, Y. et al, Agric. Bio. Chem., 35: 1068, 1971; Harrison, L. and Asakawa, Y., Phytochemistry, 26: 1211, 1987) and flavonoids (Kariyone, T., et al., 78: 1010, 1958), etc.
The present inventors have searched a novel therapeutic agent for hyperlipidemia and atherosclerosis with less side effects, from natural resources. And the present inventors have completed this invention by confirming that T. nucifera extracts or abietane diterpenoid compounds or terpenoid compounds isolated from the same has excellent anti-oxidative activity to LDL and inhibiting activity to ACAT enzyme as well.
[Disclosure]
[Technical Problem]
It is an object of the present invention to provide a novel abietane diterpenoid compound.
It is another object of the present invention to provide a composition for the prevention and the treatment of cardiovascular disease containing extracts of T. nucifera or abietane diterpenoid compounds or terpenoid compounds isolated from the same as an effective ingredient.
[Best Mode]
The present invention provides a novel abietane diterpenoid compound and a composition for the prevention and the treatment of cardiovascular disease containing extracts of T. nucifera or abietane diterpenoid compounds or terpenoid compounds isolated from the same as an effective ingredient.
The composition of the present invention includes pharmaceutical compositions, which are effective for the prevention and the treatment of cardiovascular disease, and for health food compositions.
Hereinafter, the present invention is described in detail.
The present invention provides a novel abietane diterpenoid compound represented by the following formula 1.

(R is dimetoxymethyl.)
The abietane diterpenoid compound of formula 1 is 12-hydroxyabietic-8,11,13-trien-18-dimethylacetyl.
The present invention also provides a composition for the prevention and the treatment of cardiovascular disease comprising either T. nucifera extracts or abietane diterpenoid compounds represented by the following formula 1 that are isolated from the extracts or terpenoid compounds selected from a group consisting of compounds represented by the following formula 2˜formula 5 that is also isolated from the extracts, as an effective ingredient.

(R is methyl, hydroxymethyl, aldehyde, methylester methoxymethyl.)

Abietane diterpenoid compounds of the formula 1 include ferruginol (R=methyl), 18-hydroxyferruginol (R=hydroxymethyl), 18-oxoferruginol (R=aldehyde) and 12-hydroxyabietic-8,11,13-trien-18-oic acid methyl ester (R=methyl ester). The compounds of formula 2 and formula 3 are isopimaric acid and dehydroabietinol, respectively, both are abietane diterpenoid compounds, and the compound of formula 4 is kayadiol, a labdane diterpenoid compound. The compound of formula 5 is δ-cadinol, a sesquiterpenoid compound.
The compounds of formula 1˜formula 5 can be used in the form of pharmaceutically acceptable salts, in which all the salts, hydrates and solvates that can be prepared by the conventional method are included.
T. nucifera extracts of the present invention are extracted from leaves, stems or seeds of T. nucifera by using water, alcohol or the mixture thereof. At this time, alcohol is preferably selected from a group consisting of methanol, ethanol and butanol.
Abietane diterpenoid compounds or terpenoid compounds are isolated from T. nucifera extracts by the conventional method, and reagents on the market can be used.
The extraction, separation and purification methods of T. nucifera extracts or abietane diterpenoid compounds or terpenoid compounds separated from the extracts are described hereinafter.
Dried T. nucifera leaves (stems or seeds) are dipped in water, which are heated at 40˜120° C. for 2˜24 hours. The solution is filtered to obtain extracts and solid residues. The obtained extracts are concentrated under reduced pressure to give hot water extracts of T. nucifera leaves (stems or seeds).
Or, dried T. nucifera leaves (stems or seeds) are dipped in methanol (or ethanol) for 3 weeks and then the solution is filtered. Charcoal is added to the filtrate, followed by stirring at room temperature for 12 hours. The solution is filtered again and then concentrated, to which water is added to suspend the solution. The solution is then filtered once again. The obtained upper layer is dissolved in ethyl acetate, which is then concentrated, resulting in oily yellow substances. The concentrated solution is dissolved in dichloromethane, to which n-hexane is slowly added in order to perform recrystallization. The solution is filtered with a filter-glass. The resultant liquid is concentrated, resulting in oily substances.
The prepared oil fraction was examined to measure its anti-oxidative activity to LDL and inhibiting effects on human ACAT-1 and -2, resulting in the confirmation that the oil fraction has dual inhibiting effect against LDL-oxidation and ACAT.
The obtained ethyl acetate oil fraction is separated by silica-gel column chromatography by using the mixed solvents of n-hexane and ethyl acetate as the mobile phases. At this time, the oil fraction is separated into 17 fractions (fraction 1˜17) by using the mixed solvents of n-hexane and ethyl acetate as mobile phase solvents, and at this time, the mixing ratios of n-hexane and ethyl acetate (EtOAc) are 98:2, 97:3, 95:5, 10:1, 5:1, 3:1, 1:1 and EtOAc 100% (v/v).
Fraction 8 (593 mg) that shows the best anti-oxidative activity is separated by silica-gel column chromatography by using the mixed solvents of n-hexane and ethyl acetate as mobile phases. At this time, as the mobile phase solvents, n-hexane is mixed with EtOAc at the ratios of 98:2, 95:5, 10:1, 5:1, 3:1, 1:1 and EtOAc 100% (v/v), resulting in 11 fractions (fraction 8˜8-17). Among 11 fractions, fraction 8-8˜8-10 (n-hexane:EtOAc=5:1˜1:1, 149 mg), that show the high anti-oxidative activities, are mixed, and the mixture is purified by preparative TLC (CHCl3/MeOH=80:1) and sephadex LH-20 column (CHCl3/MeOH=1:1) to give two purified compounds. These compounds are 12-hydroxyabietic-8,11,13-trien-18-dimethylacetal (R=dimetoxymethyl, 18 mg) and 12-hydroxyabietic-8,11,13-trien-18-oic acid methylester (R=methylester, 17.5 mg) of formula 1.
Ethyl acetate oil fraction obtained above is separated by silica-gel column chromatography by using the mixed solvents of n-hexane and ethyl acetate as the mobile phases. At this time, the mobile phase solvents are preferably ethyl acetate:n-hexane=10˜20:90˜80 (v/v). Among compounds of formula 1, which are pure active ingredients, ferruginol (R=methyl, 22 mg), 18-hydroxyferruginol (R=hydroxymethyl, 307 mg) and 18-oxoferruginol (R=aldehyde, 62 mg) are obtained from 1 kg of dried T. nucifera leaves by the above method.
Ethyl acetate oil fraction obtained above is separated by silica-gel column chromatography by using the mixed solvents of ethyl acetate and n-hexane as the mobile phases. The mobile phase solvents are prepared by mixing n-hexane and ethyl acetate at the ratio of 10:1, 5:1, 3:1, 1:1 (v/v) and EtOAc 100%, resulting in the separation of 11 fractions (fraction 1˜11).
Fraction 5 showing a very strong ACAT inhibiting activity is separated by silica-gel column chromatography by using the mixed solvent of n-hexane and ethyl acetate as a mobile phase. At this time, the mobile phase solvent is prepared as n-hexane:ethyl acetate=7:1 (v/v) to separate 7 fractions (fraction 5-1˜5-7) from fraction 5. Fraction 5-4 having excellent ACAT inhibiting activity is separated by silica-gel column chromatography. And, 4 fractions (fraction 5-4-1˜5-4-4) are separated therefrom by using chloroform 100% as a mobile phase. From the fraction 5-4-1, the compound of formula 2 (isopimaric acid, 76 mg), a pure active compound, is separated.
14 fractions (fraction 10-1˜10-14) are separated from fraction 10, prepared from the first column, by silica-gel column chromatography using methylene chloride:methanol=50:1 (v/v) as a mobile phase solvent. And, 11 fractions (fraction 10-4-1˜10-4-11) are separated from the active fraction 10-4 by C18 reversed phase column chromatography using methanol:water=15:1 as a mobile phase solvent. 5 fractions (10-4-5-1˜10-4-5-5) are separated from the fraction 10-4-5 having strong ACAT inhibiting activity by silica-gel column chromatography using the mixed solvents of n-hexane and ethyl acetate which are prepared at the ratios of 50:1, 30:1, 10:1, 1:1 (v/v) and EtOAc 100% as the mobile phase solvents. A pure active compound represented by formula 3 (dehydroabietinol, 25 mg) is obtained from the fraction 10-4-5-2.
A pure active compound of formula 4 (kayadiol, 40 mg) is given from the fraction 11 obtained from the first column by using recrystallization solvent n-hexane:ethyl acetate=5:1 (v/v).
12 fractions (fraction 10-6-1˜10-6-12) are separated from fraction 10-6, one of active fraction among 14 fractions obtained from the second column, by C18 reversed phase column chromatography using methanol:water=10:1 (v/v) as a mobile phase solvent. Silica-gel column chromatography is also performed with active fraction 10-6-3 using n-hexane:ethyl acetate=15:1, 10:1, 5:1, 1:1 and EtOAc 100% as the mobile phase solvents, resulting in 5 fractions (fraction 10-6-3-1˜10-6-3-5). Finally, a pure active compound of formula 5 (δ-cadinol, 15 mg) is prepared from the fraction 10-6-3-1.
T. nucifera extracts of the present invention or abietane diterpenoid compound isolated from the same has low IC50 value, indicating that it has excellent anti-oxidative activity to LDL.
T. nucifera extracts of the present invention or abietane diterpenoid compound or terpenoid compound isolated from the same show excellent ACAT inhibiting activity in hACAT-1 and in hACAT-2.
T. nucifera extracts of the present invention also reduce serum LDL and blood cholesterol.
Therefore, the composition of the present invention can be effectively used for the prevention and the treatment of cardiovascular diseases such as hyperlipidemia and atherosclerosis which are caused by the synthesis and the accumulation of cholesteryl ester.
The composition of the present invention can additionally include, in addition to T. nucifera extracts or abietane diterpenoid compound or terpenoid compound isolated from the same, one or more effective ingredients having same or similar function to the extract or compounds separated therefrom.
The composition of the present invention can also include, in addition to the above-mentioned effective ingredients, one or more pharmaceutically acceptable carriers for the administration. Pharmaceutically acceptable carriers can be selected or be prepared by mixing more than one ingredients selected from a group consisting of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrose solution, glycerol and ethanol. Other general additives such as anti-oxidative agent, buffer solution, bacteriostatic agent, etc, can be added. In order to prepare injectable solutions, pills, capsules, granules or tablets, diluents, dispersing agents, surfactants, binders and lubricants can be additionally added. The composition of the present invention can further be prepared in suitable forms for each diseases or according to ingredients by following the method represented in Remington's Pharmaceutical Science (the newest edition), Mack Publishing Company, Easton PA.
The composition of the present invention can be administered orally or parenterally (for example, intravenous, hypodermic, local or peritoneal injection). The effective dosage of the composition can be determined according to weight, age, gender, health condition, diet, administration frequency, administration method, excretion and severity of a disease. The dosage of T. nucifera extracts is 10˜2,000 mg/kg per day, and preferably 50˜500 mg/kg per day. The dosage of the compounds of formula 1˜formula 5 is 0.1˜100 mg/kg per day and preferably 0.5˜10 mg/kg per day. Administration frequency is once a day or preferably a few times a day.
T. nucifera extracts of the present invention or abietane diterpenoid compound or terpenoid compound isolated from the same was orally administered to mice to investigate toxicity. As a result, it was evaluated to be safe substance since its estimated LD50 value is much greater than 1,000 mg/kg in mice.
The composition of the present invention can be administered singly or treated along with surgical operation, hormone therapy, chemotherapy and biological reaction regulator, to prevent and treat cardiovascular diseases.
The composition of the present invention can be included in health foods for the purpose of improving cardiovascular diseases. At this time, T. nucifera extracts of the present invention or abietane diterpenoid compound or terpenoid compound isolated from the same can be added as it is or after being mixed with other food or ingredients, according to the conventional method. The mixing ratio of effective ingredients is determined by the purpose of use (prevention, health or therapeutic treatment). In the case of producing food or beverages containing T. nucifera extracts of the present invention or abietane diterpenoid compound or terpenoid compound isolated from the same, it is preferably added by 1˜20 weight %, more preferably 5˜10 weight %, to the raw material. However, the content of the extract might be less than the above when it is administered for long-term to improve health conditions but the effective dosage could contain more than the above amount because the extract of the invention is very safe.
There is no limit in applicable food, which is exemplified by meat, sausages, bread, chocolate, candies, snacks, cookies, pizza, ramyun, noodles, dairy products including ice cream, soups, beverages, tea, drinks, alcoholic drinks and vitamin complex, etc, and in fact every health food generally produced are all included.
Health beverages containing the composition of the present invention can additionally include various flavors or natural carbohydrates, etc, like other beverages. The natural carbohydrates above can be one of monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. As a sweetener, either natural sweetener such as thaumatin and stevia extract or artificial sweetener such as saccharin and aspartame can be used. The ratio of natural carbohydrate to the composition of the present invention is preferably 0.01˜0.04 g to 100 ml, more preferably 0.02˜0.03 g to 100 ml.
In addition to the ingredients mentioned above, the composition of the present invention can include in variety of nutrients, vitamines, electrolytes, flavoring agents, colouring agents, pectic acid and its salts, arginic acid and its salts, organic acid, protective colloidal viscosifiers, pH regulators, stabilizers, antiseptics, glycerin, alcohols, carbonators which used to be added to soda, etc. The composition of the present invention can also include natural fruit juice, fruit beverages and fruit flesh addable to vegetable beverages. All the mentioned ingredients can be added singly or together. The mixing ratio of those ingredients does not matter in fact, but in general, each can be added by 0.01˜0.1 weight part per 100 weight part of the composition of the invention.